Oliveira Igor Gustavo Carvalho, de Souza Israel Donizeti, Grecco Caroline Fernandes, Tumas Vitor, Queiroz Maria Eugênia Costa
Department of Chemistry, Chromatography Laboratory, Faculty of Philosophy, Science and Letters at Ribeirão Preto, University of São Paulo, 14040-901, Brazil.
Department of Neurosciences and Behavioral Sciences, Ribeirão Preto Medical School, University of São Paulo, 14040-901, Brazil.
Anal Chim Acta. 2025 Oct 1;1369:344367. doi: 10.1016/j.aca.2025.344367. Epub 2025 Jun 25.
High circulating concentrations of homocysteine (Hcy), a sulfur-containing amino acid, and homocysteic acid (HCA), an Hcy oxidized derivative, are an independent risk factor for developing Alzheimer's disease (AD), a neurodegenerative disorder that causes progressive cognitive decline. Therefore, these two endogenous compounds might be potential AD biomarkers. Nevertheless, few studies have attempted to quantify Hcy and HCA in the cerebrospinal fluid (CSF), the best validated fluid for evaluating neurodegenerative disorders.
Here, a novel restricted access-anion exchange organic monolithic phase was synthetized and used as extractive phase during in-tube SPME to analyze Hcy and HCA in CSF samples by LC-MS/MS. The innovative monolithic sorbent is porous and permeable, bears anion exchange groups that selectively extract the target analytes, and displays hydrophilic groups that restrict the access and exclude up to 90 % of macromolecules. Optimization of the synthesis parameters and evaluation of the in-tube SPME parameters improved the monolithic phase efficiency in extracting Hcy and HCA. Validation of the proposed method showed that it is linear from 8 to 250 ng mL Hcy and from 5 to 150 ng mL HCA, has adequate accuracy and precision, no significant matrix effect, and prevents carryover. Application of this innovative method to analyze CSF samples obtained from patients with AD, patients with mild cognitive impairment (MCI), or healthy controls revealed elevated Hcy levels in AD and MCI patients, with considerable discriminatory potential.
This is the first organic monolith with restricted access-anion exchange properties to be developed for in-tube SPME application. Furthermore, the proposed method addresses the need of a robust microextraction method for the determination of Hcy and HCA in cerebrospinal fluid samples. The findings also suggest Hcy as a potential AD biomarker, motivating further studies on the role this endogenous compound plays in early diagnosis and disease progression.
高循环浓度的同型半胱氨酸（Hcy，一种含硫氨基酸）和同型半胱氨酸酸（HCA，Hcy的氧化衍生物）是患阿尔茨海默病（AD）的独立危险因素，AD是一种导致进行性认知衰退的神经退行性疾病。因此，这两种内源性化合物可能是潜在的AD生物标志物。然而，很少有研究尝试对脑脊液（CSF）中的Hcy和HCA进行定量，CSF是评估神经退行性疾病最有效的液体。
在此，合成了一种新型的限进阴离子交换有机整体柱，并在管内固相微萃取（in-tube SPME）过程中用作萃取相，通过液相色谱-串联质谱（LC-MS/MS）分析脑脊液样本中的Hcy和HCA。这种创新的整体吸附剂具有多孔性和渗透性，带有选择性萃取目标分析物的阴离子交换基团，并显示出限制大分子进入并排除高达90%大分子的亲水基团。合成参数的优化和管内SPME参数的评估提高了整体柱萃取Hcy和HCA的效率。所提出方法的验证表明，其在8至250 ng/mL的Hcy和5至150 ng/mL的HCA范围内呈线性，具有足够的准确度和精密度，无显著基质效应，并可防止残留。将这种创新方法应用于分析从AD患者、轻度认知障碍（MCI）患者或健康对照者获得的脑脊液样本，结果显示AD和MCI患者的Hcy水平升高，具有相当大的鉴别潜力。
这是首个为管内SPME应用开发的具有限进阴离子交换特性的有机整体柱。此外，所提出的方法满足了对用于测定脑脊液样本中Hcy和HCA的强大微萃取方法的需求。研究结果还表明Hcy是一种潜在的AD生物标志物，促使人们进一步研究这种内源性化合物在早期诊断和疾病进展中所起的作用。
